Therapeutic nucleosides

ABSTRACT

The present invention relates to 6-substituted purine carbocyclic nucleosides and their use in medical therapy particularly in the treatment of HIV and HBV infections. The invention also relates to pharmaceutical formulations and processes for the preparation of compounds according to the invention.

This is a continuation-in-part of application Ser. No. 455,201 filedDec. 22, 1989 which is a continuation-in-part of Ser. No. 371,870 filedJune 26, 1989 now abandoned.

The present invention relates to purine nucleoside analogues containingan unsaturated carbocyclic ring in place of the sugar residue,pharmaceutically acceptable derivatives thereof, and their use inmedical therapy, particularly for the treatment of certain viralinfections.

AIDS (acquired immunodeficiency syndrome) is an immunosupressive orimmunodestructive disease that predisposes subjects to fatalopportunistic infections. Characteristically, AIDS is associated with aprogressive depletion of T-cells, especially the helper-inducer subsetbearing the OKT⁴ surface marker.

Human immodeficiency virus (HIV) has been reproducibly isolated frompatients with AIDS or with the symptoms that precede AIDS. HIV iscytopathic and appears to preferentially infect and destroy T-cellsbearing the OKT⁴ marker, and it is now generally recognized that HIV isthe etiological agent of AIDS.

Since the discovery that HIV is the etiological agent of AIDS, numerousproposals have been made for anti-HIV chemotherapeutic agents that maybe effective in treating AIDS sufferers. Thus, for example, U.S. Pat.No. 4,724,232 describes 3'-azido-3'-deoxythymidine (which has theapproved name zidovudine), its pharmaceutically acceptable derivativesand their use in the treatment of human retrovirus infections includingAIDS and associated clinical conditions. Vince et al., AntiviralResearch, 9 (1/2), 120 (1988) describes certain carbocyclic nucleosideanalogs and their use against HIV. At the Second InternationalConference on Antiviral Research, Williamsburg, Va., Apr. 10-14, 1988,(±)-9-(cis-4-(hydroxymethyl)-2-cyclopentenyl) guanine (NSC-614846), alsoknown as carbovir, was disclosed.

Worldwide, hepatitis B virus (HBV) is another viral pathogen of majorconsequence. It is most common in Asian countries, and prevalent insub-Saharan Africa. The virus is etiologically associated with primaryhepatocellular carcinoma.

The United States currently contains an estimated pool of 500,000-1million infectious carriers. Chronic active hepatitis will develop inover 25% of carriers, and often progresses to cirrhosis. It is estimatedthat 5000 people die from HBV-related cirrhosis each year in the USA,and that perhaps 1000 die from HBV-related liver cancer. Even when auniversal HBV vaccine is in place, the need for effective anti-HBVcompounds will continue. The large reservoir of persistently infectedcarriers, estimated at 220 million worldwide, will receive no benefitfrom vaccination and will continue at high risk for HBV-induced liverdisease. This carrier population serves as the source of infection ofsusceptible individuals perpetuating the incidence of diseaseparticularly in endemic areas or high risk groups such as IV drugabusers and homosexuals. Thus, there is a great need for effectiveantiviral agents, both to control the chronic infection and reduceprogression to hepatocellular carcinoma.

Clinical effects of infection with the HBV virus range from headache,fever, malaise, nausea, vomiting, anorexia and abdominal pains.Replication of the virus is usually controlled by the immune response,with a course of recovery lasting weeks or months in humans, butinfection may be more severe leading to persistent chronic liver diseaseas outlined above.

European Patent Specification No. 349242 discloses certain 6-substitutedpurine carbocyclic nucleosides and their use in medical therapyparticularly in the treatment of HIV and HBV infections. Among suchnucleosides are the compounds (±)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol and(±)-cis-4-[2-amino-6-(cyclopropylmethylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,i.e. each in the form of a racemic mixture of their relevantenantiomers.

We have now found that the individual isolated enantiomers of the twocompounds mentioned above and their pharmaceutical derivatives haveadvantageous antiviral activity, particularly against HIV and HBVinfections, coupled with low cytotoxicity and/or are useful asintermediates for the preparation of compounds having such activity.

According to one feature of the present invention there are providedenantiomeric compounds of the general formula ##STR1## (wherein Rrepresents a cyclopropylamino or N-cyclopropyl-N-methyl amino group andA represents the 2-cyclopentene-1-methanol-4-yl group in either the(1S,4R) or (1R,4S) configuration) and their derivatives (for example,esters, salts and salts of esters), the said compounds and theirderivatives each being in the form of an enantiomer substantially free(for example to the extent of less than 10% w/w, preferably less than 5%w/w) of the corresponding enantiomer.

It will be appreciated that the compounds of formula (I) comprise thecompounds having the following configurations: ##STR2## (wherein R is asdefined above).

The enantiomeric compounds of formula (I), i.e. substantially free ofthe corresponding enantiomer, thus comprise:

1)(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

2)(1R,4S)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

3)(1S,4R)-cis-4-[2-amino-6-(N-cyclopropyl-N-methylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

4)(1R,4S)-cis-4-[2-amino-6-(N-cyclopropyl-N-methylamino)-9-H-purin-9-yl]-2-cyclopentene-1-methanol

Compounds 1) and 3) above, hereinafter referred to as the (1S,4R)enantiomeric compounds of formula (I), with a negative (-) opticalrotation, have been found to have especially potent activity against HIVand HBV infections, and these compounds and their pharmaceuticallyacceptable derivatives represent preferred embodiments of the presentinvention. The compounds have the further advantage that, uponadministration, they are capable of penetrating the blood-brain barrierto provide high levels of the compounds or active metabolites thereof inthe central nervous system where manifestations of HIV infections areparticularly debilitating. Compound 1) above is especially preferred inview of its exceptionally potent activity against HIV and HBVinfections. The compound has also been found to have significantly lowertoxicity against bone marrow progenitor cells that3'-azido-3'-deoxythymidine (zidovudine) referred to above.

We have further found that phosphate derivatives of compounds 2) and 4)above, hereinafter referred to as the (1R,4S) enantiomeric compounds offormula (I), with a positive (+) optical rotation, have potent activityagainst viral infections such as those referred to above. Thesephosphate derivatives thus represent a further preferred embodiment ofthe present invention.

The reference herein to "phosphate derivatives" of the (1R, 4S)enantiomeric compounds of formula (I) denotes derivatives in which aphosphate grouping is attached to the 1-methanol group of formula (I)and includes mono-, di- and tri-phosphates.

The parent (1R,4S) enantiomeric compounds of formula (I) andnon-phosphate derivatives thereof are useful as intermediates for thepreparation of the said phosphate derivatives.

The above (1S, 4R) enantiomeric compounds of formula (I) and theirpharmaceutically acceptable derivatives, and the phosphate derivativesof the (1R, 4S) enantiomeric compounds of formula (I), are hereinafterreferred to as the antiviral compounds according to the invention.

According to further features of the present invention we provide:

a) antiviral compounds according to the invention for use in medicaltherapy particularly for the treatment of a retroviral infection or ahepatitis B viral infection; b) a method for the treatment of retroviralinfections and hepatitis B infections in a subject, e.g. a mammal suchas a human, which comprises treating the subject with a therapeuticallyeffective amount of an antiviral compound according to the invention;and c) use of an antiviral compound according to the invention in themanufacture of a medicament for the treatment of any of theabove-mentioned infections or conditions.

Examples of retroviral infections which may be treated in accordancewith the invention include human retroviral infections such as humanimmodeficiency virus (HIV), HIV-1, HIV-2 and human T-cell lymphotropicvirus (HLTV), e.g. HTLV-I or HTLV-II infections. The antiviral compoundsaccording to the invention are especially useful for the treatment ofAIDS and related clinical conditions such as AIDS-related complex (ARC),progressive generalised lymphadenopathy (PGL), AIDS-related neurologicalconditions, such as multiple sclerosis or tropical paraparesis, andanti-HIV antibody-positive and HIV-positive conditions for example inasymptomatic patients, and thrombocytopenic purpura. The compounds mayalso be used in the treatment or prevention of psoriasis.

By "a pharmaceutically acceptable derivative" in relation to the (1S,4R)enantiomeric compounds of formula (I) is meant any pharmaceuticallyacceptable salt, ester or salt of such ester, of a (1S, 4R) enantiomericcompounds of formula (I), or any other compound which, uponadministration to the recipient, is capable of providing (directly orindirectly) such an enantiomeric compound, or an antivirally activemetabolite or residue thereof.

Preferred esters of the (1S,4R) enantiomeric compounds of formula (I)include carboxylic acid esters in which the non-carbonyl moiety of theester grouping is selected from straight or branched chain alkyl, e.g.n-propyl, t-butyl, n-butyl, alkoxyalkyl (e.g. methoxymethyl), aralkyl(e.g. benzyl), aryloxyalkyl (e.g. phenoxymethyl), aryl (e.g. phenyloptionally substituted by halogen, C₁₋₄ alkyl or C₁₋₄ alkoxy or amino);sulfonate esters such as alkyl- or aralkylsulfonyl (e.g.methanesulfonyl); amino acid esters (e.g. L-valyl or L-isoleucyl); andmono-, di- or tri-phosphate esters.

The phosphate esters of compounds of formula (I), may be furtheresterified by, for example, a C₁₋₂₀ alcohol or reactive derivativethereof, or by a 2,3-di(C₆₋₂₄)acyl glycerol, for example2,3-bis-(hexanoyloxy)propyl hydrogen phosphate and 2,3-bits-(hexadecanyloxy)propyl hydrogen phosphate derivatives. In addition to such furtheresterified phosphate derivatives of the compounds of formula (I), thepresent invention further includes such derivatives of the racemiccompounds of formula (I).

With regard to the above-described esters, unless otherwise specified,any alkyl moiety present advantageously contains 1 to 18 carbon atoms,particularly 1 to 4 carbon atoms. Any aryl moiety present in such estersadvantageously comprises a phenyl group.

Pharmaceutically acceptable acid addition salts of the (1S,4R)enantiomeric compounds of formula (I) include mono- or di- basic saltswith the appropriate acid for example organic carboxylic acids such asacetic, lactic, tartaric, malic, isethionic, lactobionic and succinicacids; organic sulfonic acids such as methanesulfonic, ethanesulfonic,benzenesulfonic and p-toluenesulfonic acids and inorganic acids such ashydrochloric, sulfuric, phosphoric and sulfamic acids. The hydrochloricacid salts (i.e. the mono-and di-hydrochlorides) are particularlypreferred.

The above antiviral compounds according to the invention may be employedin combination with other therapeutic agents for the treatment of theabove infections or condition. Examples of such further therapeuticagents include agents that are effective for the treatment of viralinfections or associated conditions such as 3'-azido-3'-deoxythymidine(zidovudine), 2',3'-dideoxynucleosides such as 2',3'-dideoxycytidine,2',3'-dideoxyadenoside and 2',3'-dideoxyinosine, acyclic nucleosides(e.g. acyclovir), interferons such as α-interferon, renal excretioninhibitors such as probenicid, nucleoside transport inhibitors such asdipyridamole, dilazep, mio-, lido- or soluflazine, or hexobendine,immunomodulators such as interleukin II and granulocyte macrophagecolony stimulating factors, soluble CD₄ or genetically engineeredderivatives thereof, and phosphonoformic acid. The component compoundsof such combination therapy may be administered simultaneously, ineither separate or combined formulations, or at different times, e.g.sequentially such that a combined effect is achieved.

The antiviral compounds according to the invention, also referred toherein as the active ingredient(s), may be administered for therapy byany suitable route including oral, rectal, nasal, topical (includingbuccal and sublingual), vaginal and parenteral (including subcutaneous,intramuscular, intravenous and intradermal). It will be appreciated thatthe preferred route will vary with the condition and age of therecipient, the nature of the infection and the chosen active ingredient.

In general a suitable dose for each of the above-mentioned conditions(e.g. AIDS) will be in the range of 3.0 to 120 mg per kilogram bodyweight of the recipient (e.g. a human) per day, preferably in the rangeof 6 to 90 mg per kilogram body weight per day and most preferably inthe range 15 to 60 mg per kilogram body weight per day. The desired doseis preferably presented as two, three, four, five, six or more sub-dosesadministered at appropriate intervals throughtout the day. Thesesub-doses may be administered in unit dosage forms, for example,containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably50 to 700 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 1 to about 75μM, preferably about 2 to 50 μM, most preferably about 3 to 30 μM. Thismay be achieved, for example, by the intravenous injection of a 0.1 to5% solution of the active ingredient, optionally in saline, or orallyadministered as a bolus containing about 1 to about 100 mg/kg of theactive ingredient. Desirable blood levels may be maintained by acontinuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.4 to about 15 mg/kg of theactive ingredient.

While it is possible for the active ingredient to be administered aloneit is preferable to present it as a pharmaceutical formulation. Theformulations of the present invention comprise at least one activeingredient, as defined above, together with at least onepharmaceutically acceptable carrier or excipient. Formulations includethose adapted for oral, rectal, nasal, topical (including buccal andsublingual), vaginal or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. Suchmethods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then if necessaryshaping the product.

Formulations of the present invention adapted for oral administrationmay be presented as discrete units such as capsules or tablets eachcontaining a predetermined amount of the active ingredient; as a powderor granules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented as a bolus,electuary or paste.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxpropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent.

Moulded tablets may be made by moulding in a suitable machine a mixtureof the powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile. Tablets may optionally beprovided with an enteric coating, to provide release in parts of the gutother than the stomach.

Formulations adapted for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations adapted for rectal administration may be presented as asuppository with a suitable base comprising for example cocoa butter ora salicylate.

Formulations adapted for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

Formulations adapted for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be present inunit-dose or multidose sealed containers, for example, ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately, prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents.

The present invention further includes the following process for thepreparation of enantiomeric compounds of formula (I) above andderivatives thereof. The enantiomeric starting materials and precursorsfor such materials which are employed as described below in relation tothe process are each in the form of an anantiomer substantially free(e.g. to the extent referred to above in relation to compounds offormula (I) of the other enantiomer. The process according to theinvention comprises either:

A) treating an enantiomeric compound of formula (II): ##STR3## (whereinA is as hereinbefore defined and Z represents a precursor group for thesaid R group as defined in formula (I)) or a derivative thereof with anagent or under conditions serving to convert the precursor Z group tothe desired R group; or

B) reacting an enantiomeric compound of formula (III): ##STR4## (whereinA and R are as hereinbefore defined, R² represents hydrogen or a formylgroup and R³ represents as amino protecting group, e.g. an acyl groupsuch as a C₁₋₆ alkanoyl group, e.g. formyl, acetyl or isobutyryl) or aderivative thereof with an agent serving to effect formation of theimidazole ring in the desired compound of formula (I) followed byremoval of the R³ amino protecting group; or C) reacting an enantiomericcompound of formula (IV): ##STR5## (wherein A and R are as hereinbeforedefined and R³ is an amino protecting group, e.g. as described above inrelation to formula (III)) or a derivative thereof with an agent servingto effect removal of the R³ amino protecting group, and optionallyeffecting one or both of the following conversions in any desired order:

i) where a compound of formula (I) is formed, converting the saidcompound to a derivative thereof; or

II) where a derivative of a compound of formula (I) is formed,converting the said derivative to the parent compound of formula (I) orto a further such derivative.

Process A) may be carried out in conventional manner, for example, bytreatment of a compound of formula (II) in which Z represents a leavinggroup (e.g. a halo such as a chloro group) with an appropriate amine,i.e. cyclopropylamine or N-cyclopropyl-N-methylamine, preferably in anexcess to introduce the amino R group as defined above, advantageouslyat reflux or at a temperature greater than 50° C., preferably in thepresence of an organic solvent, for example methanol or ethanol.

Process B) may be carried out, for example, by reacting a compound offormula (III) with formic acid or a reactive formic acid derivative(e.g. triethylorthoformate or diethoxymethyl acetate) optionally with aco-solvent such as dimethylacetamide or dimethylformamide at an elevatedtemperature, preferably at 75°-90° C. This reaction is convenientlyeffected by the addition of slightly more than one equivalent of astrong anhydrous acid, e.g. with 1.1 equivalents of ethanesulfonic acidper equivalent of compound of formula (III), in which case lowertemperatures (e.g. 25° C. ) are used.

Process C) may be carried out, for example, by reacting an enantiomericcompound of formula (IV) with an acidic agent, for example, diluteaqueous hydrochloric acid.

The compounds of formula (II) employed as starting materials in processA) may be prepared for example, in an analogous manner to process B),i.e. by reacting a corresponding enantiomeric compound of formula (V)##STR6## (wherein A, Z, R² and R³ are as hereinbefore defined) or aderivative thereof with an agent serving to effect formation of theimidazole ring in the desired compound of formula (II) and to effectremoval of the R³ amino protecting group. The reaction may be carriedout using those agents and conditions described above for process B).

The compounds of formula (III) employed as starting materials in processB) may be prepared for example by treating an enantiomeric compound offormula (V) above with an agent or conditions serving to convert theprecursor group Z to the desired R group, in an analogous manner to thatdescribed for process A).

The compounds of formula (IV) referred to above may be prepared, forexample, by reacting an enantiomeric compound of formula (VI) ##STR7##(wherein A, Z and R³ are hereinbefore defined) with an agent or underconditions serving to convert the precursor group Z to the desired Rgroup, i.e. in an analogous manner to that described for process A).

The compounds of formula (VI) above may be prepared for example byreacting an enantiomeric compound of formula (V) above with an agentserving to effect formation of the imidazole ring in the desiredcompound of formula (VI), for example by treatment with formic acid or areactive formic acid derivative, as described above in relation toprocess B).

Enantiomeric compounds of formulae (II), (III), (IV), (V) and (VI) aboverepresent further features of the present invention, especially those inwhich R² represents a formyl group and/or R³ represents a C₁₋₆ alkanoylgroup, particularly acetyl or isobutyryl, and/or Z represents a halosuch as a chloro group.

Particularly preferred intermediates for the preparation of (1S,4R)-cis-4-[2-amino-6-cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,i.e. the preferred compound 1) above, include:

a)(1R,4S)-cis-N-[6-(cyclopropylamino)-9-(4-(hydroxymethyl)-2-cyclopenten-1-yl)-9H-purin-2-yl]isobutyramide;

b)(1R,4S)-cis-N-[4-chloro-5-formamido-6-((4-(hydroxymethyl)-2-cyclopentene-1-yl)amino)-2-pyrimidinyl]isobutyramide;

c)(1R,4S)-cis-N-[4-chloro-5-formamido-6-((4-(hydroxymethyl)-2-cyclopentene-1-yl)amino)-2-pyrimidinyl]acetamide;

d)(1S,4R)-cis-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol.

e) (1R,4S)-cis-N-[6-chloro-9-(4-(hydroxymethyl)-2-cyclopentene-1-yl)-9H-purin-2-yl]isobutyramide.

The enantiomeric compounds of formula (V) employed as starting materialsas described above may be prepared for example by reacting a compound offormula (VII) ##STR8## (wherein Z, R² and R³ are as hereinbefore definedand R⁴ represents a leaving group, e.g. a halo such as a chloro group)or a derivative thereof with an enantiomeric compound of formula (VIIIA)or (VIIIB) ##STR9## or a derivative thereof.

The last-mentioned reaction is advantageously effected in the presenceof a base such as a tertiary amine for example triethylamine ortrimethylamine advantageously in a organic solvent such asdimethoxyethane or ethanol.

The compounds of formula (VIIIA) or (VIIIB) having the appropriateenantiomeric configuration can be obtained by complexing thecorresponding racemic compound, i.e.(±)-4-amino-2-cyclopenene-1-methanol with an optically active carboxylicacid (for example dibenzoyl-D-tartaric acid) and then fractionalcrystallization of resulting diastereomeric salts. Alternatively,enzymatic resolution may be employed as described for example in J. Med.Chem., 1987, 30, 745 and J. Med. Chem., 1985, 28, 1385.

The enantiomeric compounds of formula (VIIIA) or (VIIIB) and theirderivatives, particularly slats thereof with optically active carboxylicacids such as dibenzoyl-D-tartaric acid, for example(1S,4R)-4-amino-2-cyclopentene-1-methanol and its dibenzoyl D tartaraterepresent a further feature of the present invention.

The compounds of formula (VII) employed as starting materials above maybe prepared in a conventional manner for example by reducing a compoundof formula (IX) ##STR10## (wherein Z, R³ and R⁴ are as hereinbeforedefined) to effect conversion of the NO₂ group to an NH₂ group andoptionally converting the resulting NH₂ group to a formamido group, forexample, by treatment with formic acid/acetic anhydride.

The compounds of formula (IX) may be prepared in conventional manner.Those compounds in which Z represents a halo, for example chloro groupmay be prepared for example by halogenating, for example usingphosphorus oxychloride, a corresponding compound of formula (X)##STR11## (wherein R³ and R⁴ are as hereinbefore defined). The compoundsof formula (X) may also be prepared in conventional manner, for exampleby reaction of a compound of formula (XI) ##STR12## (wherein R⁴ is ashereinbefore defined) with an appropriate agent serving to introduce theamino protecting group, for example by reaction with an appropriatecarboxylic acid or a functional equivalent thereof, e.g. isobutyricanhydride. The compound of formula (XI) may be prepared by nitration ofa corresponding compound of formula (XII) ##STR13## (wherein R⁴ is ashereinbefore defined).

The compounds of formulae (VII), (IX), (X) and (XI) represent furtherfeatures of the present invention particularly those in which Zrepresents a halo such as a chloro group, and/or R³ represents a C₁₋₆alkanoyl group, especially acetyl or isobutyryl, and/or R⁴ represents ahalo such as a chloro group.

Particularly preferred compounds of formulae (VII), (IX) and (X)according to the invention include:

N-(4,6-dichloro-5-formamido-2-pyrimidinyl)isobutyramide;

N-(4,6-dichloro-5-nitro-2-pyrimidinyl)isobutyramide; and

N-(4-chloro-1,6-dihydro-5-nitro-6-oxo-2-pyrimidinyl)isobutyramide.

A compound of formula (I) may generally be converted into an esterthereof by reaction with an appropriate esterifying agent, e.g. an acidhalide or anhydride. The compound of formula (I), including estersthereof, may be converted into salts thereof in conventional manner, forexample, by treatment with an appropriate acid. An ester or salt of acompound of formula (I) may be converted into the parent compound, forexample by hydrolysis.

Thus the O-monophosphate of a compound of formula (I) may be prepared bytreating the parent with an appropriate phosphorylating agent, e.g.phosphorus oxychloride as in M. Yoshikawa, T. Kato and T. Takenishi,Bulletin Chem. Soc. Japan, 1969, 42, 3505. The corresponding to )-di-andO- triphosphates may be prepared by methods described in "NucleotideAnalogs" K. H. Sheit, John Wiley and Sons, New York 1980, pp. 211-215,and D. E. Hoard and D. G. Ott, J. Amer. Chem. Soc. 1965, 87, 1785, e.g.by making the imidizolate derivative of the relevant O-monophosphate andby subsequent reaction of this derivative with phosphate to giveO-diphosphate or with pyrophosphate to give O-triphosphate. For thepreparation of esterified phosphate derivatives referred to above, theparent compound of formula (I) may be treated with an appropriatedi-alkanoyl phosphatidyl choline derivative in the presence of anappropriate phospholipase for example phospholipase D, as described inS. Shuto et al., , Tet. Lett., 28, 199, (1987) and Chem. Pharm. Bull.,36, 5020, (1988) or by reaction of a compound of formula (I) with anappropriate phosphorylating agent such as phosphorus oxychloridefollowed by work-up with an appropriate alcohol as described in A.Rosowsky & S. Kin, Nucleic Acid Chemistry, Part 3, L. B. Townsend & R.S. Tipson (Editors), John Wiley & Sons, New York, 1986, 255.

The enantiomers of the compounds of formula (I) may be resolved orisolated in conventional manner, e.g. by chromatographic separation ofdiastereomeric esters prepared by acylation of the hydroxyl on thecyclopentenyl moiety with appropriate optically active carboxylic acidderivaties as, e.g., with naproxen (J. Org. Chem. 1986, 51, 1287).

The following Examples are intended for illustration only and are notintended to limit the scope of the invention in any way. In the Examplesthe optical rotations were assigned with respect to the sodium D line(589 nm) at 20° C. The term `active ingredient` as used in Examples A toG means an antiviral compound according to the invention, especiallycompound 1) above.

EXAMPLE 1(±)-cis-4-[(2-Amino-4-chloro-6-pyrimidinyl)amino]-2-cyclopentene-1-methanol

cis-4-Acetamidocyclopent-2-enemethyl acetate [U.S. Pat. No. 4,268,672](14.88 g, 0.073 mol) and barium hydroxide octahydrate (46.19 g, 0.146mol) were refluxed in water (300 mL) under nitrogen for 18 hours. Theresulting solution was neutralized with carbon dioxide. The precipitatewas washed with water, then ethanol. The combined filtrate-wash wasevaporated to a syrup (11.16 g) which was condensed with2-amino-4,6-dichloropyrimidine (23.91 g, 0.146 mol) and triethylamine(30.5 mL, 0.219 mol) in refluxing 1-butanol (100 mL) for 1.5 hours.After addition of 1N NaOH (73 mL), the resulting mixture was evaporatedto dryness and the residual solid slurried in CHCl₂ (200 mL). Unreacted2-amino-4,6-dichloropyrimidine was filtered off and washed withchloroform (100 mL). The chloroform filtrate-wash was concentrated andchromatographed on a silica gel column. Additional pyrimidine startingmaterial was eluted with 2.5% methanol-chloroform. The title compoundwas eluted with 3.5% methanol-chloroform as an off-white solid foam(15.90 g, 91%).

¹ H-NMR: (Me₂ SO-d₆) δ 1.15-1.28 and 2.26-2.41 (2 m, 2, CH₂); 2.60-2.71(m, 1, 1'-H); 3.4 (m overlapping H₂ O, CH₂ OH); 4.625 (t, J=5.3, 1, CH₂OH); 4.95 (br s, 1, CH--N); 5.67-5.87 (m, 2, CH═CH); 6.38 (br s, 1,NH₂); 7.12 (br s, 1, NH); MS (CI) M+1, 241, 243.

Anal. Calcd. for C₁₀ H₁₃ N₄ OCl.0.2H₂ O: C, 48.99; H, 5.55; N, 22.85;Cl, 14.46. Found: C. 49.10; H, 5,57; N, 22.81; Cl, 14.40.

EXAMPLE 2(±)-cis-4-[[2-Amino-6-chloro-5-[(4-chlorophenyl)azo]-4-pyrimidinyl]-amino]-2-cyclopentene-1-methanol

(±)-cis-4-[(2-Amino-4-chloro-6-pyrimidinyl)amino]-2-cyclopentene-1-methanolfrom Example 1 (11.58 g, 48.1 mmol) and sodium acetate trihydrate (97 g)were dissolved in glacial acetic acid (225 mL) and water (225 mL). Acold solution (0°-5° C.) of 4-chlorobenzenediazonium chloride wasprepared form 4-chloroaniline (6.74 g, 52.8 mmol), concentratedhydrochloric acid (14.7 mL) water (52 mL), and sodium nitrite (4.01 g,58.2 mmol in 47 mL of water). This cold solution was added dropwise over5 minutes to the first solution. The resulting yellow precipitate wasfiltered after 18 hours, washed with water, and extracted with ethanolto give title compound as dark yellow powder (12.56 g, 69%), m.p.218°-22° C. dec.

¹ H-NMR: (Me₂ SO-d₆) δ 10.25 (d, 1, NH); 7.69 and 7.54 (both, d, J=8.9,C₆ H₄) overlapping 7.6 (br, 6, NH₂); 5.80-5.95 (m, 2, CH═CH); 5.24 (m,1, CHN); 4.75 (t, 1, CH₂ OH); 3.41 (t, 2, CH₂ OH); 2.75 (m, 1, CH); 2.41(m, 1, CH); 1.44-1.53 (m, 1, CH).

Anal. Calcd. for C₁₆ H₁₆ N₆ Cl₂ O: C, 50.67; H, 4.25; N, 22.16 Cl,18.70. Found: C, 50.89; H, 4,29; N, 22.10; Cl, 18.66.

EXAMPLE 3(±)-cis-4-[(2,5-Diamino-4-chloro-6-pyrimidinyl)-amino]-2-cyclopentene-1-methanol

The title compound of Example 2 (11.67 g) was suspended in ethanol (235mL), glacial acetic acid (30 mL), and water 235 mL. The mixture washeated to reflux under nitrogen. Zinc dust (13.5 g) was added in smallportions over 30 minutes during which time the compound dissolved. Thereaction was heated an additional 20 minutes, and then the excess zincwas filtered of from the hot solution, and it was washed with ethanol.The filtrates were evaporated, and the residue was purified on a silicagel column eluting with chloroform (1 L) and chloroform:methanol/4:1(1.8 L). The fractions containing the product were combined, and thesolvent was removed under reduced pressure to give the title compound asa red-orange oil (11.2 g,>100% yield). A pure sample was obtained duringanother small scale reaction to obtain the product as a light yellowsolid in a 76% yield.

¹ H-NMR: (Me₂ SO-d₆) δ 1.29 and 2.39 (m, 2, CH₂); 2.69 (t, 1, 1'--H);3.37 (d, 2, CH₂ OH); 3.91 (br, 2, NH₂); 4.60 (br, 1, CH₂ OH); 5.02 (m,1, CHNH); 5.56 (br s, 2, NH₂); 5.74 (m, 1, ═CH); 5.86 (m, 1, ═CH); 6.36(d, 1, CHNH).

EXAMPLE 4

(±)-cis-4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol

The title compound of Example 3 (about 9.7 g) was dissolved indiethoxymethyl acetate (100 g), and refluxed for two days. The solventwas removed under high vacuum at 50° C., and dioxane (40 mL) and 0.5NHCl (60 mL) was added. The reaction was stirred at room temperature for1.25 hours, and then chilled. The reaction was neutralized to pH 7 withcold 5N sodium hydroxide, and then it was extracted withchloroform:methanol/3:1 several times. The organic layers were driedwith magnesium sulphate, filtered, and evaporated. The residue waspurified by chromatography on a silica gel column, eluting with 2%MeOH-CHCl₃ to give 3.7 g (46% yield) of the title compound, m.p.138°-139° C.

¹ H-NMR: (Me₂ SO-d₆) δ 1.63 and 2.61 (m, 2, CH₂); 2.87 (m, 1, 1'-H);3.44 (d, 2, CH₂ OH); 5.44 (m, 1, CH-N); 5.89 (m, 1, ═CH); 6.14 (m, 1,═CH); 6.82 (br s, 2, NH₂); 8.02 (s, 1, 8-H); (CH₂ OH not seen under H₂ Opeak). UV: pH 1 λmax 315 (ξ 7370); 218 (26200); λ sh MS (EI) 265,267 (m)(CI) 266,268 (m+1).

Anal. Calcd. for C₁₁ H₁₂ N₅ ClO.2H₂ O: C, 43.79, H, 5.35; N, 23.21; Cl,11,75. Found: C, 43.67; H, 5.29; N, 23.05; Cl, 11.70.

EXAMPLE 5(±)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purine-9-yl]-2-cyclopentene-1-methanol

The title compound of Example 4 (0.50 g) was dissolved in ethanol (40mL), and cyclopropylamine (0.65 mL, 5 equivalent) was added. Thereaction was refluxed under nitrogen for 6 hours. An additional 0.65 mLof cyclopropylamine was added, and the reaction refluxed for anadditional 5.5 hours. The solvents were evaporated, and chloroform (25mL) and saturated sodium bicarbonate solution (5 mL) was added. Theaqueous layer was extracted several times with CHCl₃ to obtain the crudeproduct. This was purified on a silica gel column eluting with 3%methanol-ethyl acetate to give 0.43 g (80%) of(±-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol.This was recrystallized from acetonitrile to give 0.30 g of whitepowder; m.p. collapses at 93°-130° C.; melts at 165° C.

¹ H-NMR: (Me₂ SO-d₆) δ 0.56 and 0.63 (2, 4, 2-cyclopropyl CH₂); 1.56 and2.60 (2, 2, cyclopentenyl-CH₂); 2.85 (m, 1, 1'-H): 3.02 (m, 1,cyclopropyl CH-NH); 3.43 (m, 2, CH₂ OH); 4.71 (t, 1, CH₂ OH); 5.40 (m,1, 4'-H); 5.77 (s, 2, NH₂), overlapping 5.84 (m, 1, ═CH₂); 6.09 (m, 1,═CH); 7.23 (d, 1, NH--CH); 7.58 (s, 1, purine-8-H); ms (CI) 287 (m+1).UV: pH 1: λmax 296 (ξ 14000), 255 (10700); pH 7.0: λmax 284 (15900); 259(9200); pH 13 λmax 284 (15800 ), 259 (9100).

Anal. Calcd. for C₁₄ H₁₈ N₆ O.0.25H₂ O: C, 57.82; H, 6.41; N, 28.90.Found: C, 57.84; H, 6.45; N, 28.86.

EXAMPLE 6(±)-cis-4-(2-Amino-6-(cyclopropylmethylamino)-9H-purin-9yl)-2-cyclopentene-1-methanol

(±)-cis-4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol(0.53 g, 2 mmol) form Example 4, N-methyl-N-cyclopropylamine (KarlIndustries, Aurora, OH; 0.8477 g, 12 mmol) and methanol (20 mL) wereplaced in a Parr bomb and heated to 62° C. for 5 hours. The solution wasconcentrated and then diluted with ethanol before being brought to pH 12by the addition of 1.0N NaOH. This solution was concentrated and theresidue was purified by elution from a silica gel column with 3%methanol-chloroform (0.547 g, 91.2%). Crystallization of such a samplefrom water-ethanol yielded a white powder, m.p. 130°-131° C.

¹ H-NMR: (Me₂ SO-d₆) δ 7.61 (s, 1H, purine H-8), 6.10 (m, 1H, CH═), 5.84(m, 1H, CH═), 5.7 (br s, 2H, NH₂), 5.40 (m, 1H, CHN), (br t, 1H, OH),3.43 (m, 2H, CH₂ OH) 3.24 (br s, 4H, CH₃, NCH cyclopropyl), 2.85 (m, 1H,CH), 2.66-2.57 and 1.61-1.51 (m, 2, cyclopentenyl CH₂), 0.90-0.65 (m,4H, 2CH₂ of cyclopropyl).

Anal. Calcd. C₁₅ H₂₀ N₆ O.0,5H₂ O: C, 58.24; H, 6.84; N, 27.16. Found:C, 58.15; H, 6.86; N, 27.14.

EXAMPLE 7

(-)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

The title compound of Example 5 (0.600 g, 2.00 mmol) was dissolved in1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (Aldrich, 12 mL).Phosphoryl chloride (0.76 mL, 8.0 mmol) was added to the stirred, cooled(-10° C.) solution. After 3 minutes, cold water (100 mL) was added andthe resulting solution neutralized with 3M ammonium hydroxide. Theneutralized solution was diluted to 1 liter with water and applied to a2.5×20 cm column of DEAE Sephadex A25 (Pharmacia) which had beenpreequilibrated with 50 mM ammonium bicarbonate. The column was firstwashed with 4 liters of 50 mM ammonium bicarbonate. The O-monophosphateof(±)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolwas then eluted with a 2-liter gradient of 50 to 300 mM ammoniumbicarbonate. The fractions containing nucleotide (i.e. the aboveO-monophosphate) were evaporated to a white powder to remove ammoniumbicarbonate; 71% calculated by UV absorbance; one peak by HPLC (seebelow). Snake venom 5'-nucleotidase (EC 3.1.3.5) from Crotalus atrox(1000 IU, Sigma) was added to 1.4 mmoles of the nucleotide dissolved inwater (20 ml). The solution was incubated at 37° C. for 22 hours, atwhich time additional enzyme (1000 IU) was added. Incubation wascontinued for another 3 days. HPLC analysis (0.4×10 cm Whatman Partisisl10 strong anion exchange column; elution with a gradient of 20 mM to 1Mammonium phosphate, pH 5.5, containing 5% methanol; UV detection at 284nM) at this point showed that 50% of the starting nucleotide had beendephosphorylated to the parent nucleoside. This mixture was againapplied to a DEAE Sephadex column of the type described above. Elultionwith 4 liters of 50 mM ammonium bicarbonate gave fractions containingthe title compound. Evaporation of the water left white powder. Thismaterial was further purified by chromatography on silica gel withMeOH:CHCl₃ /1:9 to give colorless glass. The glass was solidified inacetonitrile to give (-)-cis-4-[2-amion-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol as white gummy solidwhich was dried to a solid foam at 0.5 mm Hg at 68° C. (260 mg, 86% fromracemate); ¹ H-NMR in DMSO-d₆ and mass spectrum identical with those ofthe racemate (title compound of Example 5); [α]²⁰ _(D) ⁻ 59.7°, [α]²⁰₄₃₆ ⁻ 127.8°, [α]²⁰ ₃₆₅ ⁻ 218.1°, (c=0.15, methanol).

Anal. Calcd. for C₁₄ H₁₈ N₆ O-0.8H₂ O: C,55.91; H, 6.57; N, 27.94.Found: C, 56.05; H, 6.65; N, 27.88.

Continued elution of the last-mentioned Sephadex column with a 2-litergradient of 50 to 300 mM ammonium bicarbonate gave the O-monophosphate(of the (+) enantiomer corresponding to the title compound) which wasstable to 5'-nucleotidase; the preparation of this monophosphate isdescribed in more detail in Example 9.

EXAMPLE 8(-)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolO-monophosphate

The title compound of Example 7 ((0.35 g, 1.2 mmol) was dissolved in1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (Aldrich, 5 mL).Phosphoryl chloride (Aldrich, 0.43 mL, 4.6 mmol) was added to thestirred, cooled (-10° C.) solution. After 3 minutes, cold water (20 mL)was added and the resulting solution neutralized with 3M ammoniumhydroxide. Ion exchange chromatography as described in Example 7 gavethe nucleotide as the diammonium salt after evaporation of water, whitepowder (95% yield, quantitated by UV); HPLC analysis as in Example 7shows one peak; UV λmax nM (0.1M HCl):254, 297; pH 7 phosphatebuffer):259, 284; (0.1M NaOH):259, 284. The base/phosphate ratio was1.0/1.3 as determined by the method of B. Ames (Methods in Enzymology8:115, 1966). [α]²⁰ _(D) ⁻ 69.9°, [α]²⁰ ₅₇₈ ⁻ 73.0°, [α]²⁰ .sub. 546⁻84.0° (c=0.52, MeOH: H₂ O/4:1).

EXAMPLE 9(+)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopenten-1-methanolO-monophosphate

Elution of the DEAE Sephadex column described in Example 7 after5'-nucleotidase incubation with a 2-liter gradient of 50 to 300 mMammonium bicarbonate gave nucleotide-containing fractions which, afterevaporation of water, gave the title compound as the diammonium salt;white powder (56% from title compound of Example 5); HPLC analysis as inExample 7 shows one peak; UV λ_(max) nM (0.1M HCl): 254, 297; (pH 7phosphate buffer): 259, 284; (0.1M NaOH): 259, 284. The base/phosphateratio was 1.0/0.98. [α]²⁰ _(D) ⁺ 62.0°, [α]²⁰ ₅₇₈ ⁺ 65.2°, [α]²⁰ ₅₄₆ ⁺75.0°, (c=0.54, MeOH:H₂ O/4:1).

EXAMPLE 10(+)-cis-4-[2-Amino-6-(cyclopropylamino)9H-purin-9-yl]-2-cyclopentene-1-methanol

The title compound of Example 9 (0.67 mmole) was dissolved in water (20mL) and alkaline phosphatase (EC 3.1.3.1) form calf intestine (3000 IU,Boehringer Mannheim) was added. The solution was incubated at 37° C. for19 hours, at which point HPLC analysis as described in Example 7 showedthat all of the nucleotide had been dephosphorylated. The solution wasevaporated to dryness and the residual solids extracted with refluxingethanol (100 mL). The ethanol-soluble material was adsorbed on silicagel and applied to a silica gel column. Title compound was eluted withmethanol:chloroform/1:9. Evaporation of an acetonitrile-ethanol solutiongave white solid foam (164 mg, 79%); ¹ H-NMR in DMSO-d₆ and massspectrum identical with those of the racemate (title compound of Example5); [α]²⁰ _(D) +58.7°, [α]²⁰ ₄₃₆ +126.2°, [α]²⁰ ₃₆₅ 217.5°, (c=0.10,methanol).

Anal. Calcd. for C₁₄ H₁₈ N₆ O-0.60H₂ O-0.15 EtOH: C, 56.49, H, 6.66; N,27.64. Found: C, 56.60; H, 6.63; N, 27.55.

EXAMPLE 11(-)-cis-4-[2-Amino-6-(cyclopropylmethylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

The title compound of Example 6, (200 g, 6.50 mmol) was dissolved in1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pryimidinone (Aldrich, 20 mL).Phosphoryl chloride (2.28 mL, 24.0 mmol) was added to the stirred,cooled (-10° C.) solution. After 3 minutes, cold water (80 mL) wasadded. The solution was extracted with chloroform (3×80 mL). The aqueouslayer was diluted with ethanol (400 mL) and the pH adjusted to 6 withsaturated aqueous NaOH. The precipitated inorganic salts were filteredoff. The filtrate was further diluted with ethanol to a volume of 1liter and the pH adjusted to 8 with additional NaOH. The resultingprecipitate was filtered and dried to give the O-monophosphate of(±)-cis-4-[2-amino-6-(cyclopropylmethylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolas white powder (4.0 mmoles, 62% quantitated by UV absorbance); HPLCanalysis as in Example 7 shows one peak. This racemic O-monophosphatewas dissolved in water ( 200 mL) and snake venom 5'-nucleotidase (EC3.1.3.5) from Crotalus atrox (5,000 IU, Sigma) was added. Afterincubation at 37° C. for 10 days, HPLC analysis as described in Example7 showed that 50% of the starting nucleotide had been dephosphorylatedto the nucleoside. These were separated on a 5×14 cm column of DEAESephadex A25 (Pharmacia) which had been preequilibrated with 50 mMammonium bicarbonate. The title compound was eluted with 2 liters of 50mM ammonium bicarbonate. Evaporation of water gave white powder whichwas dissolved in methanol, adsorbed on silica gel, and applied to asilica gel column. Title compound was eluted withmethanol:chloroform/1:9 as a colorless glass. An acetonitrile solutionwas evaporated to give white solid foam, dried at 0.3 mm Hg over P₂ O₅ ;649 mg (72% from racemate); ¹ H-NMR in DMSO-d₆ and mass spectrumidentical with those of the racemate (title compound of Example 6); [α]²⁰ _(D) -48.0°, [α]²⁰ ₃₆₅ -149° (c=0.14, methanol).

Anal. Calcd. for C₁₅ H₂₀ N₆ 0-0.10 CH₃ CN: C, 59.96; H, 6.72; N, 28.06.Found: C, 59.93; H, 6.76; N, 28.03.

Continued elution of the Sephadex column with 2 liters of 100 mMammonium bicarbonate and then with 2 liters of 200 mM ammoniumbicarbonate gave O-monophosphate of the (+) enantiomer corresponding tothe title compound, which was stable to 5'-nucleotidase.

EXAMPLE 12(±)-cis-4-[2-Amino-6-(cyclopropylmethylamimo)-9H-purin-9-yl]-2-cyclo-pentene-1-methanol

The fractions containing O-monophosphate of the (+) enantiomer elutedfrom the Sephadex column of Example 11 were combined and alkalinephosphatase (EC 3.1.3.1) from calf intestine (4800 IU, BoehringerMannheim) was added. The solution was incubated at 25° C. for 18 hours,at which point HPLC analysis showed that all of the nucleotide has beendephosphorylated. The solution was evaporated to dryness and theresidual solids extracted with refluxing ethanol (100 mL). Theethanol-soluble material was adsorbed on silica gel and applied to asilica gel column. Title compound was eluted with methanol:chloroform/1:9 as a colorless glass. An acetonitrile solution wasevaporated to give white solid foam, dried at 0.3 mm Hg over P₂ O₅ ; 659mg (73% from racemate); ¹ H-NMR in DMSO-d₆ and mass spectrum identicalwith those of the racemate (title compound of Example 6); [α]²⁰ _(D)+47.0°, [α]²⁰ ₄₃₆ +93.0°, [α]²⁰ ₃₆₅ +141.3° (c=0.11, methanol).

Anal. Calcd. for C₁₅ H₂₀ N₆ 0-0.1 CH₃ CN: C, 59.95; H, 6.72; N, 28.06.Found: C, 59.92; H, 6.80; N, 27.96.

EXAMPLE 13 (1S,4R)-4-Amino-2-cyclopentene-1-methanoldibenzoyl-D-tartrate

(±)-cis-4-Acetamidocyclopent-2-enemethyl acetate [U.S. Pat. No.4,268,672] (14.88 g, 0.073 mol) and barium hydroxide octahydrate (46.19g), 0.146 mol) were refluxed in water (300 ml) under nitrogen for 18hours. The resulting solution was neutralised with carbon dioxide. Theprecipitate was washed with water, then ethanol. The combined filtratewas evaporated to a syrup, (acetic acid salt of(±)-4-amino-2-cyclopentene-1-methanol) which converted to free amine bystirring with an excess of Amberlite IRA-400 (OH⁻) resin in water. Theresin was filtered off, washed with water, and the filtrate-washevaporated to a pale yellow syrup which was dried by evaporation ofportion of ethanol. Such a sample of amine (2.26 g, 20.0 mmol) anddibenzoyl-D-tartaric acid (Aldrich, 3.62 g, 10.0 mmol as 99%) weredissolved in hot absolute ethanol (35 ml). Refluxing acetonitrile (ca.150 ml) was added to the cloud point and the solution was allowed tocool slowly to room temperature. The white needles which formed wererecrystallized three times from the same solvent combination to givetitle compound as white plates (1.07 g, 37%); m.p. 160°-162°; [α]_(D) ²⁰+66.9°, [α]₄₃₆ ²⁰ +165°, [α]₃₆₅ ²⁰ +325° (c=0.28, methanol). X-raycrystallography of this salt allowed the absolute configuration of thecation to be fixed by the known configuration of the D-dibenzoyltartaric acid dianion. This salt crystallized in the space group C₂ withone C₆ H₁₂ NO cation and one-half C₁₈ H₁₄ O₈ dianion as the asymmetricunit.

Anal. Calcd. for C₆ H₁₁ NO-1/2 (C₁₈ H₁₄ O₈): C, 61.63; H, 6.21; N, 4.79.Found: C, 61.56; H, 6.24; N, 4.74.

EXAMPLE 14 (1R, 4S)-4-Amino-2-cyclopentene-1-methanoldibenzoyl-L-tartrate

This salt was formed and crystallized as described in Example 13, exceptthat dibenzoyl-L-tartaric acid was used. Three crystallizations fromethanol-acetonitrile gave the title compound as white plates (1.00 g,34%); m.p. 160°-162°; [α]_(D) ²⁰ -68.2°, [α]₄₃₆ ²⁰ -169°, [α]₃₆₅ ²⁰-333°, (c=0.24, methanol).

Anal. Calcd. for C₆ H₁₁ NO-1/2 (C₁₈ H₁₄ O₈): C, 61.63; H, 6.21; N, 4.79.Found: C, 61.59; H, 6.21; N, 4.76.

EXAMPLE 15(±)-cis-N-[4-chloro-5-formamido-6-[[4-(hydroxymethyl)-2-cyclopentene-1-yllamino]-2-pyrimidinyl]acetamide

N(5-Amino-4,6-dichloropyrimidin-2-yl)acetamide (J. Org. Chem. 1975, 40,3141) was formulated by addition of 96% formic acid (20 ml) to asolution of (0.75 g, 3.4 mmoles) dissolved in acetic anhydride (20 ml).The resulting solution was stirred at 25° C. for one hour and thenevaporated to give (0.77 g, 91%); structure confirmed by ¹ H-NMR andmass spectrum. This tan powder (840 mg, 3.37 mmol),(±)-cis-4-amino-2-cyclopentene-1-methanol (940 mg, 8.2 mmol), andtriethylamine (0.80 g, 8.0 mmol) were warmed in ethanol (50 ml) in anoil bath (70°-80° C.) under nitrogen for 50 minutes and evaporated to adark oil which was chromatographed on silica gel. Title compound waseluted with 5% methanol-chloroform as a peach-colored solid foam (840mg). Crystallization from methanol gave white granules (575 mg, 52%);m.p. 189°-193°; .sup. 1 H-NMR (DMSO-d₆) δ 10.23 (br, 1.0, NHAc), 9.3(br, 1.0 NHCHO), 8.15 and 7.90 (both s, 1.0, HC═0 from two conformers,peaks coalesce at 60° C.), 7.42 and 7.22 (both d, J=8.3, total 1.0,CH--NH from two conformers, peaks coalesce at 60° C.), 5.9 and 5.7 (bothm, 2.0, CH═CH), 5.05 (m, 1, OH), 3.39 (m, 2, CH₂ OH), 2.72 (m, 1, CH),2.40 (m, 1, 1/2 CH₂), 1.36 m, 1, 1/2 CH₂).

Anal. Calcd. for C₁₃ H₁₆ N₅ O₃ Cl: C, 47.93; H, 4.95; N, 21.50; Cl,10.88. Found: C, 47.99; H, 4.96; N, 21.42; Cl, 10.96.

EXAMPLE 16(±)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo-pentene-1-methanol

The title compound of Example 15 (0.91 g, 2.79 mmol) was dissolved indry DMF (1 ml). Triethylorthoformate (10 ml) and ethane sulfonic acid(0.29 ml, 3.4 mmol) were added and the solution heated at 65° C. for 24hours. The solution was evaporated to a syrup. The syrup was dissolvedin 1N HCl (15 ml) and stirred for three hours. The pH was adjusted to 7with 5N sodium hydroxide and the resulting mixture (oil formed) wasextracted with i-propanol:chloroform/1:3 (3×100 ml). The combinedorganic layers were dried (MgSO₄) and evaporated to a red glass (0.93g). A sodium of this glass in methanol (20 ml) was heated withcylcopropylamine (2 ml) in a Parr bomb at 70° C. for 18 hours. Theresulting solution was evaporated to a dark glass which was adsorbed onsilica gel. Elution with 7% methanol-ethylacetate gave title compound(148 mg, 19%) as white powder, after trituration with acetonitrile; ¹H-NMR (DMSO-d₆) identical with that of the title compound of Example 5.

EXAMPLE 17(±)-1R,4S)-cis-N-[4-Chloro-5-formamido-6-{[4-(hydroxymethyl)-2-cyclopentene-1-yl]amino}-2-pyrimidinyl]acetamide

(1S,4R)-4-Amino-2-cyclopentene-1-methanol dibenzoyl-D-tartrate preparedas described in Example 13 (2.76 g, 9.02 mmol) was dissolved in water(20 ml) and applied to a column of 65 ml of Amberlite IA-400 (OH⁻ form)anion exchange resin. The column was washed with water. Basic fractionswere combined and evaporated to a residual oil which was dried byevaporation of absolute ethanol and then at 0.5 mm to give oil (darkensrapidly in air) which was used immediately. This oil was dissolved inethanol (5 ml) and added to a solution ofN-(4,6-dichloro-5-formamido-2-pyrimidinyl)acetamide (2.07 g, 8.31 mmol),prepared as described in Example 15, and triethylamine (2.50 g, 24.8mmol). The resulting dark solution was heated (oil bath 75°-80° C.)under nitrogen for 50 minutes. The solution was evaporated to a syrupwhich was applied to a silica gel column. Title compound was eluted with3 to 5% methanol-chloroform as a pale yellow solid foam (1.59 g, 54%); ¹H-NMR identical with that of crystallized sample. Such a sample wascrystallized from ethanol to give white granules, m.p. 194°-195° C.; ¹H-NMR (DMSO-d₆) identical with that of the title compound of Example 15;[α]²⁰ _(D) +2.7°, [α]₅₄₆ ²⁰ +2.9°, [α]₄₃₆ ²⁰ -2.5°, [α]₃₆₅ ²⁰ -41.2°(c=0.238, methanol).

EXAMPLE 18(-)-(1S,4)-cis-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol

The title compound of Example 17 (1.15 g, 3.53 mmol) was gently refluxedin diethoxylmethyl acetate (45 ml) under nitrogen for 3.5 hours. Theresulting pale yellow solution was concentrated at 0.5 mm Hg to a yellowsyrup. The syrup was stirred in 1N HCl (50 ml) for 1.0 hour. Thissolution was neutralized with sodium bicarbonate and evaporated todryness. The residual solids were extracted with methanol and themethanol-soluble material applied to a silica gel column. Elution of thecolumn with 10% methanol-ethyl acetate gave title compound as a paleyellow solid foam (730 mg), 78%); ¹ H-NMR (DMSO-d₆): identical with thatof racemate (title compound of Example 4); [α]_(D) ²⁰ -114.9° (c=0.26,MeOH).

EXAMPLE 19(-)-(1S,4R)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

The title compound of Example 18 (560 mg, 2.11 mmol) in methanol (12 ml)was heated with cyclopropylamine (2.4 ml) in a Parr bomb at 78° C. for17 hours. The solvent was evaporated and the residue chromatographed onsilica gel. Title compound was eluted with 5-7% methanol-ethyl acetateas a colorless solid foam (367 mg, 59%); ¹ H-NMR (DMSO-d₆) identicalwith that of Example 7; [α]_(D) ²⁰ -59.0° (c=0.28, MeOH) confirms theabsolute configuration of the title compound of Example 7.

EXAMPLE 20 (1S,4R)-4-Amino-2-cyclopentene-1-methanoldibenzoyl-D-tartrate

2-Azabicyclo[2.2.1]hept-5-en-3-one [Daluge and Vince, J. Org. Chem.1978, 43, 2311 and U.S. Pat. No. 4,268,672] (44.0 g, 0.400 mole) wasstirred in 2N HCl in methanol (0.5 L) at 25° C. for 1.5 hours. Volatileswere evaporated to leave(±)-cis-methyl-4-amino-2-cyclo-pentene-1-carboxylate hydrochloride as anoff-white powder (71.1 g). Trituration of such a sample withdiethylether gave a white powder, m.p. 92.5°-95° C. [J. Org. Chem. 1981,46, 3271; m.p. 82°-83° C.]; ¹ H-NMR (DMSO-d₆) δ 8.25 (br s, 3, NH₃ ⁺),6.1 and 5.9 (both m, 2, CH═CH), 3.64 (s) overlapping 3.75-3.6 (m, total4, OMe and CH), 2.65-2.45 and 2.05-1.85 (both m, 2, CH₂).

Anal. Calcd for C_(C) ₇ H₁₁ NO₂ -HCl: C, 47.33; H, 6.81; N, 7.89; Cl,19.96. Found: C, 47.41; H, 6.84; N, 7.85; Cl, 19.89.

(±)-cis-Methyl-4-amino-2-cyclopentene-1-carboxylate hydrochloride (17.7g, 0.100 mole) and diisobutyl-aluminum hydride (0.500 mole as a 1Msolution in hexane) were refluxed in hexane (200 ml) for 6 hours. Theresulting solution was cooled and 10 ml of 1M aqueous ammonium chlorideand then methanol (200 ml) were added. This mixture was refluxed for 30minutes and MgSO₄ (10 g) added. Solids were filtered off and washed withadditional methanol. The filtrate-wash was evaporated to a dark oil(15.5 g); ¹ H-NMR (DMSO-d₆) identical to that of(±)-4-amino-2-cyclopentene-1-methanol prepared as described in Example13. Such a sample, after purification by chromatography on silica gel(EtOH:CHCl₃ :NH₄ OH/10:90:1) was crystallized with dibenzoyl-D-tartaricacid to form the title compound.

EXAMPLE 21cis-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopenten-1-yl]methylR-2,3-bis-(hexadecanoyloxy)propyl hydrogen phosphate

A solution of L-α-dipalmitoyl phosphatidyl choline (150 mg, 0.2 mmol,Sigma) in 6 ml of chloroform was added to a flask containing(±)-cis-4-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanol(300 mg, 1.03 mmol), phospholipase D, Type VII (from Streptomyces, 1.0mg, specific activity 185 units/mg, Sigma) and pH 4.5 buffer (1.5 ml,250 mM in CaCl₂, 200 mM in NaOAc adjusted to pH 4.5 by addition of 0.1NHCl). The resulting biphase was stirred at 45° C. (oil bath) for 1 hour.The layers were separated and the aqueous layer extracted withchloroform (3×6 ml). The combined organic layers were washed with 1NHCl, dried and concentrated. Such a sample was purified by elution from2 silica gel columns with 12% methanol-chloroform to yield titlecompound, 120 mg (47%). This material was solidified usingethylacetate-acetonitrile to produce a light yellow powder m.p.155°-157° C.; ¹ H-NMR (CD₃ CD-CDCl₃) δ 7.78 (s, overlapping solvent,purine H-8), 6.12 and 5.88 (m, 2,. HC═CH), 5.53 (m, 1, CHNcyclopentene), 5.22 (m, 1, CO₂ CH), 4.37 (dd, J=3, 12; 1, 0.5 POCH₂glycerol), 4.12 (m, 1, 0.5 POCH₂ glycerol), 3.42 (m, 4, OCH₂ glycerol,OCH₂), 3.11 (br m, 1, CH), 2.90 (m, 1, NCH), 2.78 (m, 1, 0.5 CH₂cyclopentene), 2.27 (m, 4, 2CH_(CO) ₂), 1.70 (m, 1, 0.5 CH₂cyclopentene), 1.56 (br m, 4, 2CH₂ CH₂ CO₂), 1.27 (br m, 38, 24 CH₂),0.88 (m, 6, 2CH₃), 0.83 (m, 2, CH₂ cyclopropyl), 0.60 (m, 2, CH₂cyclopropyl).

Anal. Calcd. for C₄₉ H₈₅ N₆ O₈ P-2.4 H₂ O: C, 61.28; H, 9.42; N, 8.75;P, 3.22. Found: C, 60.97; H, 9.12; N, 8.78; P, 2.96.

EXAMPLE 22[cis-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopenten-1-yl]methylR-2,3-bis-(hexanoyloxy)propyl hydrogen phosphate

A solution of L-α-dicaproyl phosphatidylcholine (300 mg, 0.66 mmol,Sigma) in 15 ml of CHCl₃ was added to a flask containing(±)-cis-4-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanol(378 mg, 1.32 mmol), phospholipase D, Type VII (from Streptomyces, 1.04mg, specific activity 185 units/mg, Sigma), pH 4.5 buffer (4.5 ml, 250mM in CaCl₂, 200 mM in NaOAc adjusted to pH 4.5 with HCl) and CHCl₃ (3ml). The resulting biphase was stirred at 45° C. (oil bath) for 4 hours.The layers were separated and the organic layer washed with 1N HCl (2×4ml). The combined aqueous layers were back washed with chloroform (10ml). The combined organic layers were dried (MgSO₄) and concentrated.The residue was placed on a silica gel column and the title compound waseluted with 16% methanol-chloroform and concentrated to yield a fineyellow powder. This material was dissolved in ethanol and concentrated(3×50 ml) before drying under high vacuum to yield 103 mg (21% yield) ofa light yellow powder, m.p. 182°-185° C.

¹ H-NMR: (DMSO-d₆) α 7.61 (s, 1, purine H8), 7.22 (br s, 1, NH), 6.09(m, 1, 0.5 CH═CH), 5.89 (m, overlapping br s at 5.83, 3, 0.5 CH═CH,NH₂), 5.41 (br m, 1, CHN), 5.09 (br m, 1, CO₂ CH), 4.30 (dd; J=2.7, 12;1, 0.5 POCH₂ glycerol), 4.08 (m, 1, 0.5 POCH₂ glycerol), 3.80 (br moverlapping br m at 3.75, 4, OCH₂ glycerol, OCH₂), 3.02 (br m, 2, CH,NCH cyclopropropyl), 2.65 (m, 1, 0.5 CH₂ cyclopentene), 2.23 (+, J=7.5,4, 2 CH₂ CO₂), 1.48 (br m, 5, 2 CHCH₂ CO₂, 0.5 CH₂ cyclopentene), 1.23(br m, 8, 2 (CH₂)₂), 0.84 (m, 6, 2 CH₃), 0.67 and 0.58 (m, 4, 2 CH₂cyclopropyl).

Anal. Calcd. for C₂₉ H₄₅ N₆ O₈ P-3.9 H₂ O, 0.2 CHCl₃, 0.05 EtOH: C,48.00; H, 7.33; N, 11.46; Cl, 2.9. Found: C, 48.65; H, 6.61; N, 10.81;Cl, 2.5.

The preceding example is an adaptation of the procedure by Satoshi Shutoet al. Tetrahedron Letters, Vol. 28, No. 2, pp. 199-202, 1987.

EXAMPLE 23N-(4-Chloro-1,6-dihydro-5-nitro-6-oxo-2-pyrimidinyl)isobutyramide

6-Chloro-5-nitroisocytosine (J. Chem. Soc. 1960, 5041; J. Org. Chem.1975, 40, 3141) was protected by heating the yellow solid (14.88 g,78.09 mmol) to 100° C. for one hour in isobutyric anhydride (250 ml) andconcentrated sulphuric acid (3-4 drops). The resulting solution wastreated with anhydrous methanol (100 ml), stirred at 50° C. for half anhour, concentrated to a third of the original volume, and the titlecompound (14.97 g, 74%) was collected by filtration as pale yellowcrystals; m.p. 196°-199° C. (dec); ¹ H-NMR (DMSO-d₆) δ 1.12 (d, J=6.9,Hz, 6H, (CH₃), 2.75 (m, J=6.9, Hz, 1H, (CH₃)₂ CH), 12.41 (br s, 1H).

Anal. Calcd. for C₈ H₉ N₄ O₄ Cl: C, 36.87; H, 3.48; N, 21.50; Cl, 13.60.Found: C, 36.94; H, 3.48; N, 21.44; Cl, 13.53.

EXAMPLE 24 N-(4,6-Dichloro-5-nitro-2-pyrimidinyl)isobutyramide

The title compound of Example 23 (10.0 g, 38.37 mmol) was heated toreflux in phosphorus oxychloride oxychloride (200 ml) andN,N-diethylaniline (3-4 drops) for 5 hours under nitrogen. The solutionwas then cooled to room temperature, concentrated to dryness, and thesyrup was dissolved in cold (˜-10° C.) methylene chloride (200 ml). Theorganic layer was treated with saturated aqueous sodium bicarbonate (100ml) with vigorous stirring, and the temperature was kept below 5° C. assolid sodium bicarbonate was added portionwise to elevate the pH tobetween 5 and 7. The layers were separated and the aqueous phase wasextracted with methylene chloride. The combined organic layers werefiltered over phase-separator paper, concentrated and dried under vacuumto give the title compound (7.71 g, 72%) as a yellow-white solidsufficiently pure to employ in the next step. Recrystallisation of thesolid from hexane/methylene chloride provided an analytical sample, m.p.166°-169° C.; ¹ H-NMR (DMSO-d₆) δ 1.09 (d, J=6.9 Hz, 6H, (CH₃)₂ CH),2.79 (m, J=6.9 Hz, 1H, (CH₃)₂ CH), 11.61 (s, 1H).

Anal. Calcd. for C₈ H₈ N₄ O₃ Cl₂ : C, 34.43; H, 2.89; N, 20.08; Cl,25.41. Found: C, 34.53; H, 2.89; N, 20.02; Cl, 25.40.

EXAMPLE 25 N-(4,6-Dichloro-5-formamido-2-pyrimidinyl)isobutyramide

The title compound of Example 24 (6.77 g, 24.26 mmol) was placed in aParr bottle containing 220 ml absolute EtOH and 10.0 g (wet) Raneynickel catalyst that had been previously shaken under hydrogen (40 psi)for 10 minutes. The mixture was shaken under hydrogen (40 psi) for anhour, filtered over celite, and the filtrate was concentrated to ayellow-white solid that was dried under vacuum overnight. This solid wasstirred in 1,2-dichloroethane (250 ml) at 0° C. Acetic anhydride (30 ml)was added, followed by formic acid (30 ml), dropwise under nitrogen. Theresulting mixture was stirred at room temperature for 2 hours,concentrated to half the original vlume; and azeotroped with toluene toremove residual formic/acetic acid. The crude solid was triturated withmethanol to give the title compound (4.92 g, 73%) as an off-white solid;m.p. 206°-209° C. (dec); ¹ H-NMR (DMSO-d₆) δ 1.08 (d, J=6.8 Hz, 6.0(CH₃)₂ CH), 2.74 (m, J=6.8 Hz, 1.0 (CH₃)₂ CH), 8.18 (d, J=10.3 Hz) and10.26 (br s) [total 1.0, NHCHO from two conformers], 11.17 br s, 1.0).

Anal. Calcd. for C₉ H₁₀ N₄ O₂ Cl₂ : C, 39.01; H, 3.64; N, 20.22; Cl,25.59. Found: C, 39.13; H, 3.68, N, 20.12; Cl, 25.67.

EXAMPLE 26

(±)-(1R,4S)-cis-N-[4-Chloro-5-formamido-6-{[4-(hydroxymethyl)-2-cyclopentene-1-yl]amino}-2-pyrimidinyl]isobutyramide.

(1S,4R)-4-Amino-2-cyclopentene-1-methanol dibenzoyl-D-tartrate (2.44 g,8.15 mmol) prepared as described in Example 13, was dissolved in 90%ethanol (20 ml) and the solution added to a column of Amberlite IRA-400(OH⁻) resin (30 ml) which had been prewashed with the same solvent.

Elution with 90% ethanol gave basic fractions which on concentration andevaporation of portions of toluene-ethanol, left(1S,4R)-4-amino-2-cyclopentene-1-methanol as pale yellow oil (1.4 g)which was condensed immediately withN-(4,6-dichloro-5-formamido-2-pyrimidinyl isobutyramide (2.26 g, 8.15mmol) prepared as described in Example 25, in 1,2-dimethoxyethane (100ml) with triethylamine (2.3 ml, 16.3 mmol) at 95°-110° C. for 1.5 hours.The resulting solution was evaporated to a dark yellow syrup which waschromatographed on silica gel. Elution of column with 5-7.5%methanol-chloroform gave the title compound as pale yellow solid (2.45g, 84%). Crystallisation of such a sample from acetonitrile gave thetitle compound as fine white crystals, mp. 194°-195.5° C.

¹ H-NMR (DMSO-d₆) δ 10.21 (s, 1, NHCOCHMe₂), 9.29 (s, 1, NHCHO), 8.12(s, 1, CHO), 7.18 (d, J=7.9, 1, CHNH), 5.8 and 5.7 (both m, 2, CH═CH),5.08 (m, 1, CHN), 4.71 (t, J=5.06 1, OH), 3.37 (m, 2, CH₂ OH); 2.9-2.6(m,2, CHMe₂ and CH), 2.40 (m, 1, 0.5 CH₂), 1.33 (m, 1, 0.5 CH₂); [α]_(D)²⁰ +4.4°, [α]₃₆₅ ²⁰ -20.7° (c=0.237, MeOH).

Anal. Calcd. for C₁₅ H₂₀ N₅ ClO₃ : Calcd.: C, 50.92; H, 5.70; N, 19.79;Cl, 10.02. Found: C, 50.67; H, 5.78; N, 19.62; Cl, 9.92.

EXAMPLE 27(-)-(1R,4S)-cis-N-(cyclopropylamino)-9-(4-hydroxymethyl)-2-cyclopentene-1-yl)-9H-purin-2-yl]isobutyramide

(+)-(1R,4S)-cis-N-[4-chloro-5-formamido-6-{[4-(hydroxymethyl(-2-cyclopentene-1-yl]amino}-2-pyrimidinyl]isobutyramide(1.949 g, 5.44 mmol) prepared as described in Example 26, was stirredwith triethylorthoformate (30 ml) in an ice-water bath whileconcentrated hydrochloric acid (2.0 ml) was added dropwise over twominutes. The resulting clear solution was stirred at ambient temperatureovernight.

The volatiles were removed under vacuum and the residual syrup(containing a (1R,4S)-cis-N-[6-chloro-9-(4-hydroxymethyl)-2-cyclopentene-1-yl)-9H-purin-2-yl]isobutyramideorthoester conjugate) refluxed in ethanol (30 ml) with cyclopropylamine(10 g) 2.5 hours. Evaporation left a syrup which was dissolved in 10%isopropanolchloroform (200 ml). This solution was stirred vigorouslywith saturated aqueous sodium bicarbonate (25 ml). The organic layer wasseparated and the aqueous layer washed with additional 10%isopropanol-chloroform. The combined organic layers were dried (MgSO₄).Evaporation left a pale yellow glass (2.4 g) which was chromatographedon silica gel. The title compound was eluted with 2-3% methanol-ethylacetate as a white solid (1.02 g, 53%); recrystallisation of such asample from methanol-acetonitrile gave the title compound as whiteneedles; mp. 197.5°-198°.

¹ H-NMR (DMSO-d₆) δ 9.75 (s, 1, NHCO) 7.93 (s, 1, purine H-8), 7.82 (brs, 1, NH-cyclopropyl), 6.12 and 5.92 m, 2, CH═CH), 5.50 (m, 1, CH-N,4.73 (t, J=5.3, 1, OH), 3.46 (m, 2, CH₂ -O), 3.25-3.00 (m, 2, CHMe₂ andCH), 2.91 (m, 1, CH), 2.75-2.6 (m, 1, 0.5 CH₂), 1.7-1.6 (m, 1, 0.5 CH₂),1.07 (d, J=6.8, 6, CHMe₂), 0.75-0.6 (m, 4, 2 cyclopropyl, CH₂); [α]_(D)²⁰ -70.7°, [α]₄₃₆ ²⁰ -159.0° (c=1.02, MeOH).

Anal Calcd. for C₁₈ H₂₄ N₆ O₂ : Calcd.: C, 60.66; H, 6.79; N, 23.58.Found: C, 60.62; H, 6.83; N, 23.51.

Continued elution of the column with 5% methanol-ethyl acetate gaveadditional title compound contaminated by ca. 10% of(-)-(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolas a pale yellow solid foam (928 mg).

EXAMPLE 28(-)-(1S,4R)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

(-)-(1R,4S)-cis-N-[6-(cyclopropylamino)-9-(4-hydroxymethyl)-2-cyclopentene-1-yl)-9H-purin-2-yl]isobutyramide(1.33 g, 3.73 mmol) prepared as described in Example 27, was stirredwith 1N hydrochloric acid (40 ml) for 2 days at ambient temperature. ThepH was adjusted to 7.0 with sodium hydroxide and the mixture evaporatedto dryness. The residual solids were triturated with hot EtOH (3×25 ml).The ethanol was evaporated to leave yellow glass which was chromagraphedon silica gel. The title compound was eluted with 3% methanol-ethylacetate as a colourless solid foam (857 mg, 80%), ¹ H-NMR and [α]_(D)²⁰, identical with that of the title compound of Example 19.

EXAMPLE 29 (-)-(1S,4R)-cis-4-2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolhydrochloride.

(-)-(1S,4R)-cis-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(1.90 g, ca. 6.3 mmol by ¹ H-NMR) was dissolved in 1N hydrochloric acid(7.0 ml) and ethanol. The solution was evaporated to dryness and theresidue redissolved in ethanol (15 ml). Ethyl acetate was added slowly,to a total volume of 80 ml. The off-white powder which formed wasfiltered off and dried under vacuum to give the title compound (2.07 g,97%); mp. collapses at 125°-130°, dec. above 138° C., [α]₅₈₉ ²⁰ -27.1°,[α]₄₃₆ ²⁰ -52.3° (c=0.199, MeOH).

Anal. Calcd. for C₁₄ H₁₈ N₆ O.HCl.0.8H₂ O: Calcd.: C, 49.87; H, 6.16; N,24.92; Cl, 10.51. Found: C, 49.91; H, 6.16; N, 24.95; Cl, 10.52.

EXAMPLE 30(-)-(1S,4)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolDihydrochloride.

(-)-(1S,4R)-cis-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(857 mg, 3.00 mmol) was dissolved in ethanolethyl acetate and 1Nethereal hydrochloric acid (12 ml) was added. The fine white precipitatewas washed with ethyl acetate and dried under vacuum to give the titlecompound (642 mg, 75%); mp. 176°-180° dec.

Anal. Calcd. for C₁₄ H₁₈ N₆ 0.2HCl: Calcd.: C, 46.81; H, 5.61; N, 23.39;Cl, 19.74, Found: C, 46.82; H, 5.64; N, 23.33; Cl, 19.67.

EXAMPLE 31(1R,4S)-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-diphosphate

(+)-(1R,4S)-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-monophosphate, prepared as described in Example 9, was converted tothe triethylammonium salt by taking a solution containing 0.5 mmol ofthe monophosphate as the ammonium salt, combining it with 10 ml of 0.5Mtriethylammonium bicarbonate and drying in vacuo, followed by anotheraddition of 10 ml of 0.5M triethylammonium bicarbonate, then drying.Then, three times, 10 ml of acetonitrile were added and dried in vacuo.This was dissolved in 10 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (Aldrich) then 0.43 gof 1,1'-carbonyl diimidazole (Aldrich, 2.6 mmol) was added and stirredfor 2 hours at room temperature. Methanol (0.18 ml, 4.5 mmol) was addedand stirred for 30 minutes. Tributylammonium pyrophosphate (Sigma, 1.2g, 2.6 mmol) was added, stirred for 18 hours at room temperature, then 1g of additional tributylammonium pyrophosphate (2.2 mmol) was added andstirred 8 hours at 40° C., then 50 ml of water was added. BothO-diphosphate and O-triphosphate were formed since the tributylammoniumpyrophosphate contained orthophosphate impurity.

The reaction products were separated by DEAE Sephadex ion exchangechromatography in a 2.5×18 cm column of DEAE Sephadex A25 (Pharmacia)which had been equilibrated with 50 mM of ammonium bicarbonate (ABC).The column was washed with 1 l of 50 mM ABC then with a 2 l lineargradient of 50 to 800 mM ABC to elute the title compound followed bytriphosphate, as described in more detail in Example 32. The fractionscontaining diphosphate were combined, dried in vacuo, redissolved inwater then dried again to yield the ammonium salt of the title compound(0.077 mmol, 15% yield). UV scan: in 0.1M HCl λ max=254 and 298 nm; atpH 7 λ max=259 and 284 nm: in 0.1M NaOH λ max=259 and 284 nm.

An aliquot of diphosphate was treated with alkaline phosphatase (calfintestine, Boehringer Mannheim), sampled at various times and developedon thin layer chromatography (PEI- cellulose, Brinkman, 1M LiCl/1Mformic acid 1:1). A sequential conversion of diphosphate tomonophosphate to nucleoside was observed. The final amount of -phosphate released was determined by the method of Bencini (Bencini, D.A., Wild, J. R., and O'Donovan, G. A. Analytical Biochemistry132:254-258(1983)) and the base/phosphate ratio was determined to be1.0/11.5, indicating the presence of inorganic phosphate. UV purity was99.8% on analytical HPLC (strong anion exchange column eluted with agradient of 10 mM to 1M ammonium phosphate, pH 5.5).

EXAMPLE 32(+)-(1R,4S)-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-triphosphate

Continued elution of the column described in Example 31 gave, onevaporation, the ammonium salt of the title compound. This salt wasconverted to the sodium salt by passage through a Dowex AG 50W-X8(Bio-Rad) resin column (sodium form, 20 ml). The fractions containingnucleotide were concentrated in vacuo to yield 0.31 mmol (61%). UV scan:in 0.1M HCl λmax=254 and 299 nm; at pH 7 λmax=259 and 284 nm: in 0.1MNaOH λmax=259 and 284 nm. Optical rotation in water at 3.83 g/100 ml was[α]20=+43.2° at 589 nm. UV purity was 99.1% on analytical HPLC (stronganion exchange column eluted with a gradient of 10 mM to 1M ammoniumphosphate, ph 5.5) with 0.9% diphosphate present. An aliquot oftriphosphate was treated with alkaline phosphatase (calf intestine,Boehringer Mannheim), sampled at various times and developed on thinlayer chromatography (PEI-cellulose, Brinkman, 1M LiCl/1M formic acid1:1). A sequential conversion of triphosphate to diphosphate tomonophosphate to nucleoside was observed. The final amount of phosphatereleased was determined by the method of Bencini (Bencini, D. A., Wild,J. R., and O'Donovan, G. A. Analytical Biochemistry 132:254-258 (1983))and the base/phosphate ratio was determined to be 1.0/2.7.

EXAMPLE 33(1S,4R)-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-diphosphate

The(-)-(1S,4R)-4-(2-Amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-monophosphate, prepared as described in Example 8, was converted tothe triethylammonium salt by taking a solution containing 0.49 mmol ofthe monophosphate as the ammonium salt, combining with 5 ml of 0.5Mtriethylammonium bicarbonate and drying in vacuo, followed by another 5ml of 0.5M triethylammonium bicarbonate then repeating twice. Then,three times, 5 ml of acetonitrile were added and dried in vacuo. Thiswas dissolved in 7 ml of1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (Aldrich) then 0.39 gof 1,1'-carbonyl diimidazole (Aldrich, 2.4 mmol) was added and stirredfor 30 minutes at room temperature. Methanol (0.16 ml, 4.0 mmol) wasadded stirred for 30 minutes. Tributylammonium pyrophosphate (made byexchanging the salt of tetrasodium pyrophosphate for hydrogen on anion-exchange resin, then neutralizing with tributylamine and drying, 2.4mmol) was added, stirred for 18 hours at room temperature, then 50 ml ofwater was added. Both O-diphosphate and O-triphosphate were formed sincethe tributylammonium pyrophosphate contained orthophosphate impurity.

The reaction products were separated by DEAE Sephadex ion exchangechromatography in a 2.5×18 cm column of DEAE Sephades A25 (Pharmacia)which had been equilibrated with 50 mM ammonium bicarbonate (ABC). Thecolumn was washed with 1 L of 100 mM ABC then with a 2 L linear gradientof 100 to 800 mM ABC to elute the to elute the title compound followedby the triphosphate as described in more detail in Example 34. Thefractions containing disphosphate were combined, dried in vacuo,redissolved in water then repeated twice to yield the ammonium salt ofthe title compound (0.032 mmol, 6% yield). UV scan: in 0.1M HCl λmax=254and 298 nm; at pH 7 λmax=259 and 284 nm: in 0.1M NaOH λmax=258 and 284nm.

An aliquot of triphosphate was treated with alkaline phosphatase (calfintestine, Boehringer Mannheim), sampled at various times and developedon thin layer chromatography (PEI-cellulose, Brinkman, 1M LiCl/1M formicacid 1:1). A sequential conversion of diphosphate to monophosphate tonucleoside was observed. The final amount of phosphate released wasdetermined by the method of Bencini (Bencini, D. A., Wild, J. R., andO'Donovan, G. A. Analytical Biochemistry 132:254-258 (1983)) and thebase/phosphate ratio was determined to be 1.0/4.7, indicating thepresence of inorganic phosphate. UV purity was 97% on analytical HPLC(strong anion exchange column eluted with a gradient of 10 mM to 1Mammonium phosphate, pH 5.5).

EXAMPLE 34(1S,4R)-4-(2-amino-6-(cyclopropylamino)-9H-purin-9-yl)-2-cyclopentene-1-methanolO-triphosphate

Continued elution of the column described in Example 33 gave, onevaporation, the ammonium salt of the title compound. This salt wasconverted to the sodium salt by passage through a Dowex AG 50W-X8(Bio-Rad) resin column (sodium form, 20 ml). The fractions containingnucleotide were concentrated in vacuo to yield 0.4 mmol (81%). UV scan:in 0.1M HCl λmax=254 and 299 nm; at pH 7 λmax=259 and 284 nm: in 0.1MNaOH λmax=259 and 284 nm. Optical rotation in water at 6.14 g/100 ml was[α]20=-47.1° at 589 nm. UV purity was 99.5% on analytical HPLC (stronganion exchange column eluted with a gradient of 10 mM to 1M ammoniumphosphate, ph 5.5) with 0.5% diphosphate present. An aliquot oftriphosphate was treated with alkaline phosphatase (calf intestine,Boehringer Mannheim), sampled at various times and developed on thinlayer chromatography (PEI-cellulose, Brinkman, 1M LiCl/1M formic acid1:1). A sequential conversion of triphosphate to diphosphate tomonophosphate to nucleoside was observed. The final amount of phosphatereleased was determined by the method of Bencini (Bencini, D. A., Wild,J. R., and O'Donovan, G. A. Analytical Biochemistry 132:254-258 (1983))and the base/phosphate ratio was determined to be 1.0/2.8.

EXAMPLE 35(1S,4R)-4-[2-Amino-6-(cyclopropylmethylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol

(1S,4R)-4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol(274 mg, 1.00 mmol), N-cyclopropyl-N-methylamine (0.71 g, 10 mmol), andabsolute ethanol (6 mL). The residue was chromatographed on silica gel.The Title compound was eluted with 10% methanol-chloroform as acolorless glass. Evaporation of an ethanol solution and drying withphosphorus pentoxide at 0.2 mm Hg gave the title compound as a whitesolid foam (293 mg, 98%); ¹ H-NMR and [α]²⁰ 589 identical with those ofthe title compound of Example 11.

EXAMPLE A Tablet Formulations

The following formulations A, B and C are prepared by wet granulation ofthe ingredients with a solution of povidone, followed by addition ofmagnesium stearate and compression.

Formulation A

    ______________________________________                                                         mg/tablet                                                                              mg/tablet                                           ______________________________________                                        (a)  Active ingredient 250        250                                         (b)  Lactose B.P.      210         26                                         (c)  Povidone B.P.      15         9                                          (d)  Sodium Starch Glycollate                                                                         20         12                                         (e)  Magnesium Stearate                                                                               5          3                                                                 500        300                                         ______________________________________                                    

Formulation B

    ______________________________________                                                         mg/tablet                                                                              mg/tablet                                           ______________________________________                                        (a)  Active ingredient 250        250                                         (b)  Lactose           150        --                                          (c)  Avicel PH 101      60         26                                         (d)  Povidone B.P.      15         9                                          (e)  Sodium Starch Glycollate                                                                         20         12                                         (f)  Magnesium Stearate                                                                               5          3                                                                 500        300                                         ______________________________________                                    

Formulation C

    ______________________________________                                                       mg/tablet                                                      ______________________________________                                        Active ingredient                                                                              100                                                          Lactose          200                                                          Starch            50                                                          Povidone          5                                                           Magnesium stearate                                                                              4                                                                            359                                                          ______________________________________                                    

The following formulations, D and E, are prepared by direct compressionof the admixed ingredients. The lactose in formulation E is of thedirect compression type (Dairy Crest - "Zeparox").

Formulation D

    ______________________________________                                                          mg/tablet                                                   ______________________________________                                        Active ingredient   250                                                       Pregelatinised Starch NF15                                                                        150                                                                           400                                                       ______________________________________                                    

Formulation E

    ______________________________________                                                      mg/tablet                                                       ______________________________________                                        Active ingredient                                                                             250                                                           Lactose         150                                                           Avicel          100                                                                           500                                                           ______________________________________                                    

Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients(below) with a solution of povidone followed by the addition ofmagnesium stearate and compression.

    ______________________________________                                                             mg/tablet                                                ______________________________________                                        (a)     Active ingredient  500                                                (b)     Hydroxypropylmethylcellulose                                                                     112                                                        (Methocel K4M Premium)                                                (c)     Lactose B.P.        53                                                (d)     Povidone B.P.       28                                                (e)     Magnesium Stearate  7                                                                            700                                                ______________________________________                                    

Drug release takes place over a period of about 6-8 hours and iscomplete after 12 hours.

EXAMPLE B Capsule Formulations Formulation A

A capsule formulation is prepared by admixing the ingredients ofFormulation D in Example A above and filling into a two-part hardgelatin capsule. Formulation B (infra) is prepared in a similar manner.

Formulation B

    ______________________________________                                                            mg/capsule                                                ______________________________________                                        (a)     Active ingredient 250                                                 (b)     Lactose B.P.      143                                                 (c)     Sodium Starch Glycollate                                                                         25                                                 (d)     Magnesium Stearate                                                                               2                                                                            420                                                 ______________________________________                                    

Formulation C

    ______________________________________                                                          mg/capsule                                                  ______________________________________                                        (a)       Active ingredient                                                                           250                                                   (b)       Macrogol 4000 B.P.                                                                          350                                                                           600                                                   ______________________________________                                    

Capsules of formulation C are prepared by melting the Macrogol 4000 BP,dispersion the active ingredient in the melt and filling the melt into atwo-part hard gelatin capsule.

Formulation D

    ______________________________________                                                      mg/capsule                                                      ______________________________________                                        Active ingredient                                                                             250                                                           Lecithin        100                                                           Arachis Oil     100                                                                           450                                                           ______________________________________                                    

Capsules of formulation D are prepared by dispersing the activeingredient in the lecithin and arachis oil and filling the dispersioninto soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule)

The following controlled release capsule formulation is prepared byextruding ingredients a, b and c using an extruder, followed byspheronisation of the extrudate and drying. The dried pellets are thencoated with release-controlling membrane (d) and filled into atwo-piece, hard gelatin capsule.

    ______________________________________                                                            mg/capsule                                                ______________________________________                                        (a)      Active ingredient                                                                              250                                                 (b)      Microcrystalline Cellulose                                                                     125                                                 (c)      Lactose B.P.     125                                                 (d)      Ethyl Cellulose   13                                                                           513                                                 ______________________________________                                    

EXAMPLE C Injection Formulation Formulation A

    ______________________________________                                        Active ingredient         0.200 g                                             Hydrochloric acid solution, 0.1M, or                                                                    4.0 to 7.0                                          Sodium hydroxide solution, 0.1M q.s. to pH                                    Sterile water q.s. to     10 ml                                               ______________________________________                                    

The active ingredient is dissolved in most of the water (35°-40° C.) andthe pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or thesodium hydroxide as appropriate. The batch is then made up to volumewith the water and filtered through a sterile micropore filter into asterile 10 ml amber glass vial (type 1) and sealed with sterile closuresand overseals.

Formulation B

    ______________________________________                                        Active ingredient       0.125  g                                              Sterile, pyrogen-free, pH 7                                                                           25     ml                                             phosphate buffer q.s. to                                                      ______________________________________                                    

EXAMPLE D Intramuscular Injection

    ______________________________________                                        Active ingredient    0.20      g                                              Benzyl Alcohol       0.10      g                                              Glycofurol 75        1.45      g                                              Water for Injection  q.s. to 3.00                                                                            ml                                             ______________________________________                                    

The active ingredient is dissolved in the glycofurol. The benzyl alcoholis then added and dissolved, and water added to 3 ml. The mixture isthen filtered through a sterile micropore filter and sealed in sterile 3ml amber glass vials (type 1).

EXAMPLE E Syrup

    ______________________________________                                        Active ingredient    0.25      g                                              Sorbitol Solution    1.50      g                                              Glycerol             2.00      g                                              Sodium Benzoate      0.005     g                                              Flavor, Peach 17.42.3169                                                                           0.0125    ml                                             Purified Water       q.s. to 5.00                                                                            ml                                             ______________________________________                                    

The active ingredient is dissolved in a mixture of the glycerol and mostof the purified water. An aqueous solution of the sodium benzoate isthen added to the solution, followed by addition of the sorbitolsolution and finally the flavor. The volume is made up with purifiedwater and mixed well.

EXAMPLE F Suppository

    ______________________________________                                                               mg/suppository                                         ______________________________________                                        Active ingredient         250                                                 Hard Fat, BP (Witepsol H15 - Dynamit NoBel)                                                            1770                                                                          2020                                                 ______________________________________                                    

One-fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C. maximum. The active ingredient is sifted through a 200 μm sieve andadded to the molten base with mixing, using a silverson fitted with acutting head, until a smooth dispersion is achieved. Maintaining themixture at 45° C., the remaining Witepsol H15 is added to the suspensionand stirred to ensure a homogenous mix. The entire suspension is passedthrough a 250 μm stainless steel screen and, with continuous stirring,is allowed to cool to 40° C. At a temperature of 38° C. to 40° C., 2.02g of the mixture is filled into suitable, 2 ml plastic molds. Thesuppositories are allowed to cool to room temperature.

EXAMPLE G Pessaries

    ______________________________________                                                       mg/pessary                                                     ______________________________________                                        Active ingredient                                                                              250                                                          Anhydrate Dextrose                                                                             380                                                          Potato Starch    363                                                          Magnesium Stearate                                                                              7                                                                            1000                                                         ______________________________________                                    

The above ingredients are mixed directly and pessaries prepared bydirect compression of the resulting mixture.

Antiviral Activity a) Anti-HIV Activity

(1S,4R)-cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolwas tested for anti-HIV activity in MT₄ cells according to the methoddescribed by Averett, D. R., J. Virol. Methods, 23 1989, 263-276 and wasfound to have an IC₅₀ value of 4.0±1.4 μM (average of 10determinations).

b) Anti-HBV Activity

The human HBV producer cell line of HepG2,2.2.15, described andcharacterised by Sells et al., PNAS 84: 1005, 1987 and J. Virol. 62:2836, 1988 has been shown to share many characteristics of the HBVchronically infected hepatocyte. It is infectious as demonstrated by theability to cause disease in chimpanzees.

To test compounds for anti-HBV activity, monolayer cultures were treatedwith the test compound: 50-200 μM for ten days.

Supernatant media containing extracellular virion DNA (Dane particles)were harvested on days three, six and ten, treated with proteinase K (1mg/mL) and sodium dodecyl sulfate (1%), and incubated at 50° C. for onehour. DNA was extracted with equal volumes of phenol followed bychloroform and then precipitated by ammonium acetate and propanol. TheDNA precipitate was dissolved and collected on nitrocellulose using theprocedure of Schleicher and Schuell (S&S, 10 Optical Ave., Kenne, N.H.03431, Publication #700, 1987), and treated as described by Southern, J.Mol. Biol., 98, 503, 1975. Cells were harvested, and the intracellularDNA was obtained after cell lysis with guanidine isothiocyanate. Theintracellular DNA was handled in the same manner as the extracellularDNA. After precipitation by ammonium acetate and propanol, theintracellular DNA precipitate was dissolved, cut by restrictionendonuclease, Hind III, applied to agarose gel and then treated asdescribed by Southern to determine the quantity of replicativeintermediate forms. The antiviral effect of the drug was determined bymeasuring at least a 100-fold reduction of the amount of Dane particlesextruded into the culture medium and a similar decrease in theintracellular replicative intermediates.

(1S,4R)-cis-4-[2-Amino-6-(N-cyclopropyl-N-methylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanolwas tested by the above procedure and found to have potent anti-HBVactivity at 100 μM.

I claim:
 1. Enantiomeric compounds of formula (III) ##STR14## wherein Arepresents the 2-cyclopentene-1-methanol-4-yl group in either the(1S,4R) or (1R,4S) configuration, R represents a cyclopropylamino orN-cyclopropyl-N-methylamino group, R² represents hydrogen or a formylgroup and R³ represents a C₁₋₆ alkanoyl group, or a salt or esterthereof.
 2. Enantiomeric compounds of formula (V) ##STR15## wherein Arepresents the 2-cyclopentene-1-methanol-4-yl group in either the(1S,4R) or (1R,4S) configuration, Z represents a halo group, R²represents hydrogen or a formyl group and R³ represents a C₁₋₆ alkanoylgroup, or a salt or ester thereof. 3.(1R,4S)-cis-N-[4-Chloro-5-formamido-6-((4-(hydroxymethyl)-2-cyclopentene-1-yl)amino)-2-pyrimidinyl]isobutyramidesubstantially free of the corresponding (1S,4R) enantiomer.
 4. Compoundsof formula (VII) ##STR16## wherein Z represents a halo group, R²represents hydrogen or a formyl group, R³ represents a C₁₋₆ alkanoylgroup and R⁴ represents a halo group, or a salt thereof. 5.N-(4,6-Dichloro-5-formamido-2-pyrimidinyl)isobutyramide.
 6. Compounds offormula (IX) ##STR17## wherein Z represents a halo group, R³ representsa C₁₋₆ alkanoyl group and R⁴ represents a halo group or a salt thereof.7. N-(4,6-Dichloro-5-nitro-2-pyrimidinyl)isobutyramide.
 8. Compounds offormula (X) ##STR18## wherein R³ represents a C₁₋₆ alkanoyl group and R⁴represents a halo group or a salt thereof. 9.N-(4-Chloro-1,6-dihydro-5-nitro-6-oxo-2-pyrimidinyl)isobutyramide.